Nanoparticles have attracted interest as potential bioreactors, imaging or diagnostic agents, and vehicles for drug delivery. For these applications, the nanoparticles should ideally be biocompatible, biodegradable, have substantial storage capacity for hydrophilic and hydrophobic solutes, and have tunable properties for targeting cellular structures and releasing solutes on cue (Gindy & Prud'homme, 2009, Exp. Opin. Drug Deliv. 6(8):865-78). Nanoparticles that satisfy some of these constraints include porous particles, metallic or semi-conductor nanoparticles, and vesicles. Of these constructs, vesicles hold particular promise, based on their large storage capacity.
Vesicles consist of a shell of amphiphilic molecules in a tail-to-tail configuration enclosing an aqueous solvent. The canonical structure of vesicle membranes is the bilayer, in which two layers of amphiphiles are ordered such that their hydrophobic tails are oriented inward and their hydrophilic heads are oriented outward toward both the exterior and luminal aqueous phases. Bilayer membranes surround the cells of living organisms, and are selectively permeable, only allowing specific substrates to pass into the cytoplasm.
Vesicles have great potential to be used as flexible delivery systems in vitro and in vivo. In order to be used as therapeutic or diagnostic vehicles, the vesicles may be loaded with hydrophobic or hydrophilic solutes in the membrane or lumen, respectively, and these solutes may be released upon application of specific stimuli (heat, light, or pH, for example). Vesicles could be further designed to present specific targeting moieties on their exterior surface, so that the therapeutic agent contained within the vesicles would be delivered to a specific site (such as a tumor), thereby minimizing collateral toxicity to surrounding tissue. Furthermore, proteins such as proton pumps could be incorporated into the membrane, creating a vesicular bioreactor.
The simplest way to construct a vesicle is from phospholipids and lipid blends (Evans & Needham, 1986, Faraday Disc. Chem. Soc. 81:267-80). Because phospholipids are naturally occurring components, their biocompatibility is not a major concern. Unfortunately, phospholipid vesicles tend to be mechanically weak and unable to withstand the substantial shear stresses in the circulation, and are quickly cleared by the reticulo-endothelial system (Discher et al., 2002, J. Phys. Chem. B 106(11):2848-54). Adding polyethylene glycol chains to the outer lipid shell of phospholipids extends their circulation time, but does not substantially improve their mechanical stability (Lasic & Needham, 1995, Chem. Rev. 95(8):2601-28). Additionally, lipids are rapidly oxidized and difficult to modify chemically, and exist in a narrow range of molecular weights, limiting their use as the main component of tunable vesicles.
Vesicles have been alternatively prepared from synthetic amphiphiles, such as block copolymers (polymersomes) (Discher et al., 1999, Science 284:1143-46) or amphiphilic dendrimers (dendrimerisomes) (Percec et al., 2010, Science 328(5981):1009-14). Like amphiphilic phospholipids, amphiphilic block co-polymers of the correct molecular weight and block fractions self-assemble into a lamellar phase (bilayer) in water (Discher et al., 1999, Science 284:1143-46). Polymersomes can be made from polymers with a wide variety of molecular weights (Bermudez et al., 2002, Macromol. 35:8203-08) and chemistries (Ahmed & Discher, 2004, J. Contr. Rel. 9(1):37-53; Cheng et al., 2009, Adv. Funct. Mat. 19(23):3753-59; Fraaije et al., 2005, Faraday Disc. 128:355-61; Opsteen et al., 2007, Chem. Comm. 30:3136-38; Upadhyay et al., 2009, Biomacromol. 10(10):2802-08), and thus their material properties are widely tunable. For example, polymersomes with poly(butadiene) hydrophobic cores may be made mechanically tough by crosslinking the unsaturated side chains in the membrane core (Discher et al., 2002, J. Phys. Chem. B 106(11):2848-54). Alternatively, polymersomes may be stabilized by linking together terminal acrylate groups added to the ends of polymers (Katz et al., 2009, Langmuir 25(8):4429-34). These ultra-tough vesicles are mechanically robust and have long circulation times, and may be used for stable storage of contents such as hemoglobin (for making artificial red blood cells) and imaging agents (Cheng et al., 2009, Adv. Funct. Mat. 19(23):3753-59), and these contents may be released from the vesicles in response to chemical changes (such as pH) or external stimuli (such as light) (Ghoroghchian et al., 2006, Macromol. 39(5):1673-75; Rehor et al., 2003, J. Contr. Rel. 87(1-3):246-47; Kamat et al., 2010, Adv. Funct. Mat. 20:2588-96; Robbins et al., 2009, J. Am. Chem. Soc. 131(11):3872-74)
However, polymersomes have limitations as vesicle building materials. Polymersomes are made from synthetic polymers, many of which are toxic in humans and animals. They may also be difficult to functionalize with bioactive ligands, resulting in low attachment efficiencies. Furthermore, polymers are typically polydisperse, with wide ranges of molecular weights. The polydispersity limits the precision of the structures that can be assembled from the polymers.
Nanovesicles have also been recently prepared from amphiphilic Janus dendrimers. A dendrimer is a monodisperse branched structure, wherein the number of branches and the chemistry in each branch is tunable. However, one problem in the development of dendrimer-based vesicles is the poor control in attaching ligands to the functional ends of dendrimers, which leads to heterogeneous products. Another problem is the change in polymer phase structure upon attachment of large peptides to the dendrimer. Yet another problem is that dendrimers may include synthetic chemical residues that may be toxic.
As a possible alternative approach, vesicles may be made from peptides or small proteins, which, as naturally occurring molecules, are biocompatible. In theory, proteins may be engineered to have sizes similar to those of vesicle-forming polymers. Furthermore, proteins may be engineered with exquisitely fine control (to avoid the polydispersity of polymers) and chemical diversity (using the library of naturally occurring amino acids). Specific functional groups may be genetically introduced in the peptide chain by changing the gene that codes for the protein, or may be attached by protein-ligation methods to protein molecules in the preformed vesicle.
As an example of vesicles prepared with proteins, a pH switchable vesicle was prepared from a di-block co-peptide of poly-(L-glutamic acid)-b-poly(L-lysine), E15K15 (Rodriguez-Hernandez & Lecommandoux, 2005, J. Am. Chem. Soc. 127(7):2026-27). At pH values between 5 and 9, both amino acid chains were charged, and the peptide chains were dispersed. However, at pH values below 5 or above 9, one of the two chains was neutralized, the other chain remained charged, and the peptide assembled into vesicles with an orientation dictated by pH (Deming, 2007, Prog. Pol. Sci. 32(8-9):858-75; Holowka et al., 2007, Nature Mat. 6(1):52-57; Holowka & Deming, 2010, Macromol. Biosci. 10(5):496-502; Holowka et al., 2005, J. Am. Chem. Soc. 127(35):12423-28). Likewise, poly-(L-lysine)-b-poly(L-leucine) di-block co-peptide polymers (KnLm) were synthesized, and K60L20 was shown to assemble into vesicular membranes. Peptides with other ratios of the two amino acids assembled into structures different from vesicles, such as fibrils and sheet-like membranes. Moreover, vesicles of K60L20 could be extruded through polycarbonate membranes into 100 nm vesicles, useful for biological applications (Holowka et al., 2005, J. Am. Chem. Soc. 127(35):12423-28). Vesicles from poly(L-arginine)-b-poly(L-leucine) in the same molar ratio (R60L20) were found to be internalized by HeLa cells in culture (Holowka et al., 2007, Nature Mat. 6(1):52-57). In this aspect, the peptide R60L20 mimicked TAT peptides, which are rich in arginine residues and facilitate cell internalization.
Taken together, these results demonstrate that polypeptide chains may be used to prepare vesicles, and the incorporation of specific amino acids in the peptide chain may allow control of biological activity and function of the formed vesicle structures. However, the use of peptides as building blocks of vesicles still faces challenges. Such peptides have so far been prepared by synthetic chemical methods, rather than by molecular biology, and are thus polydisperse. Chemical ligation of functional groups to the peptide terminals is often inefficient, leading to complex mixtures. Furthermore, chemical methods allow the synthesis of peptides of only moderate size, whereas yields and purities are often poor for larger target peptides.
Oleosins are plant proteins that solubilize fat bodies in plants. Oleosins have an N-terminal hydrophilic segment, followed by a hydrophobic core and another hydrophilic segment at the C-terminus (Lacey et al., 1998, Biochem. J. 334:469-77; Hsieh & Huang, 2004, Plant Physiol. 136(3):3427-34). The hydrophobic core is more conserved than the N- and C-terminal domains, which are thought to interact with the surface of oil bodies (Alexander et al., 2002, Planta 214(4):546-51; Beaudoin & Napier, 2002, Planta 215(2):293-303). Modeling studies suggested that the hydrophobic core is helical (possibly a coiled-coil) and bifurcated by a proline knot (a highly conserved stretch containing three prolines that introduces a 180° turn in the chain) (Alexander et al., 2002, Planta 214(4):546-51).
Oleosin enters the endoplasmic reticulum (ER) co-translationally, where it is introduced into the wall of a growing oil body (Beaudoin & Napier, 2000, Planta 210(3):439-45). The hydrophobic core of oleosin appears to be essential to anchoring the nascent protein in the ER; substituting leucines for the three prolines leaves the protein able to enter the ER, but unable to transfer to the oil body (Abell et al., 1997, Plant Cell 9(8):1481-93). Replacing half of the hydrophobic core of oleosin with a duplicate of the other half appears to have no effect on the ability of the protein to insert into the ER (Abell et al., 2004, Plant J. 37(4):461-70). This suggests that the degree of hydrophobicity and the length of the hydrophobic core (rather than its specific sequence) give oleosin its unique geometry in membranes. In the case of the sesame oleosin, reducing the size of the hydrophobic core by half had no effect on the ability of oleosin to stabilize artificial oil bodies (Peng et al., 2007, J. Agr. Food Chem. 55(14):5604-10). The ability of oleosins to stabilize oil bodies is also not affected by protein fusion at the termini of the protein chain (Chiang et al., 2007, Prot. Expr. Pur. 52(1):14-18). These observations have resulted in attempts to target the expression of clinically important proteins in plants to oil bodies where they may be easily harvested (Chiang et al., 2007, Prot. Expr. Pur. 52(1):14-18; Chiang et al., 2005, J. Agric. Food Chem. 53(12):4799-4804; Nykiforuk et al., 2006, Plant Biotech. J. 4(1):77-85). Oleosin fusions have also been used for protein purification in vitro (Chiang et al., 2007, Prot. Expr. Pur. 52(1):14-18).
There remains a need in the art to identify novel chemical compounds or biomolecules that may be utilized to generate vesicles useful for drug delivery or imaging techniques. The ideal chemical compound or biomolecule that forms the vesicles should be easily prepared and isolated, biocompatible, mechanically robust, and responsive to environmental conditions. The present invention fulfills these needs.